Electrophoresis band skewing

[TaO!]

I've been running electrophoresis of PCR products (~520 bases) through a 2% agarose and getting skewed bands. I tried dropping the voltage to 70V but the samples seem to be "climbing" the sides of the well. I've had a look at some trouble shooting and they suggest many different things so I was hoping that someone here might see something I dont. *Gel attached*

Ta!

TaO!

*Edit* Sorry, I just realised this should maybe have gone into the PCR forum.

[kiki06]

Hi Tao,


I think your samples may have too much salt, or your buffer may have been old/not well prepared.

Another possibility is due to bad running buffer, your agarose got hot and you got the bands like that however last time I did that it looked a bit different.

Did you purify your PCR product before running? Also how much did you load? It may be too much DNA also..

[TaO!]

I didn't purify my PCR product, however I was also thinking that it may be a buffer problem because the ladder was a professional preparation (lanes 7-9), and it shows the same skewing. I loaded 5uL of PCR product plus 4uL of 2x loading buffer into each well. I loaded 3 preparations of the Fermentas 1kb ladder to see the difference in concentration of the ladder.

Lane 7: 2uL of ladder + 4uL of 2xlb
Lane 8: 3uL of ladder + 4uL of 2xlb
Lane 9: 4uL of ladder + 4uL of 2xlb